A selective fluorescent turn-on NIR probe for cysteine

Article abstract of DOI:10.1039/c2ob07046d

A selective and sensitive turn-on fluorescent NIR probe for cysteine has been developed. Cleavage of 2,4-dinitrobenzenesulfonyl (DNBS) with thiols switches the weakly fluorescent aza-BODIPY dye (λ_em = 734 nm, Φ_f = 0.03) to a strongly

Full text of DOI:10.1039/c2ob07046d

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A selective fluorescent turn-on NIR probe for cysteine†
Xin-Dong Jiang,*a Jian Zhang,a Xiangmin Shaoa and Weili Zhao*a,b
Received 7th December 2011, Accepted 16th January 2012
DOI: 10.1039/c2ob07046d
benzo[d]oxazol-4-ylmethanamine, 1,10-phenanthrolin-5-amine
A selective and sensitive turn-on fluorescent NIR probe for
cysteine has been developed. Cleavage of 2,4-dinitrobenzene-
sulfonyl (DNBS) with thiols switches the weakly fluorescent
aza-BODIPY dye (λem = 734 nm, Φf = 0.03) to a strongly flu-
orescent species in the NIR region (λem = 755 nm, Φf = 0.14).
and others led to fluorescent probes for thiols too.14 Therefore,
these probes have great potential for applications in the detection
of thiols in vitro and in environmental studies. However, the
emission range (400–600 nm) of these probes for detecting
thiols is in the visible region. NIR fluorescent dyes can greatly
reduce background absorption, fluorescence, light scattering, and
improve the detectable sensitivity and selectivity.15 More impor-
tantly, NIR fluorescent dyes have deep penetration to achieve a
distinct image in vivo. BODIPY dyes are well-known to be
highly fluorescent, very stable, and exceptionally insensitive to
the polarity of solvents as well as to pH.16 We had reported
novel NIR aza-BODIPY dyes (λabs = 740 nm) in previous
work.17 Herein we designed a NIR probe for detecting cysteine
1 (Scheme 1, see ESI†). DNBS is a good potent electron accep-
ter and can disturb the electronic structure of the BODIPY fluoro-
phore,13b thus probe 1 is hypothesized to be weakly fluorescent
due to photoinduced electron transfer, while thiol-specific clea-
vage reaction will release the hydroxyl group, and in turn makes
the BODIPY fluorophore 2 strongly fluorescent (Scheme 1).
The spectroscopic properties of 1 in the absence and presence
of cysteine were examined. The absorption and emission
maxima of probe 1 were 717 (ε = 48 000 M−1 cm−1) and
734 nm respectively, and probe 1 was weakly fluorescent (Φf =
0.03) (the solid curve in Fig. 2). Upon addition of cysteine, the
absorption and emission maxima were red-shifted to 735 (ε =
63 000 M−1 cm−1) and 755 nm respectively, and a significant
increase in fluorescence quantum yield (Φf = 0.14) was observed
(the dashed curve in Fig. 2, Scheme 1), which is in good agree-
ment with a typical PET process.13,18 To the best of our knowl-
edge, 1 is the first example of a NIR probe for detecting thiol
Low molecular weight thiols (LMWTs) play vital roles in living
organisms.1 For example, cysteine (Cys) is an essential biologi-
cal molecule required for its detoxifying function, immunologi-
cal competence, and growth and delay of senility of cells and
tissues in living systems.1,2 Lack of cysteine causes health pro-
blems such as retarded growth, hair depigmentation, and liver
damage.3 Therefore, selective and sensitive detection of intra-
cellular cysteine has attracted increasing interest.4
Among the available techniques to detect and quantify these
thiols, luminescent methods have been extensively pursued due
to their simplicity and high sensitivity.5 This process of detecting
thiols involves a specific reaction between probes and thiols,
such as cleavage reaction by thiol,6 cyclization with aldehyde,7
metal complex-displacement coordination,8 and others.9 The
cleavage reaction by deprotection of the 2,4-dinitrobenzenesulfo-
nyl (DNBS) group was found to primarily switch a PET10 or
ICT11 process to achieve a turn-on fluorescent probe (Fig. 1). For
example, Maeda et al. reported a probe for thiols based on rho-
damine in 2005;12 Hu and Zhao subsequently reported PET
probes based on BODIPY;13 changes to the fluorophore, such as
Fig. 1 Concept of a turn-on probe based on deprotection of the
2,4-dinitrobenzenesulfonyl group.
aThe Key Laboratory for Special Functional Materials, Ministry of
Education, Henan University, Henan, 475004, China. E-mail: xdjiang@
henu.edu.cn, zhaow@henu.edu.cn; Tel: +86 0378 3881358
bSchool of Pharmacy, Fudan University, Shanghai, 201203, China.
Tel: +86 021 51980111
Scheme 1 Design of a fluorescent turn-on NIR probe 1 and cleavage
of probe 1 with thiol(s) to generate 2.
†Electronic supplementary information (ESI) available: Experimental
details and 1H, 13C NMR spectra. See DOI: 10.1039/c2ob07046d
1966 | Org. Biomol. Chem., 2012, 10, 1966–1968
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Fig. 4 Fluorescence emission–time profile of probe 1 towards cysteine.
The fluorescence intensity of 20 μM probe 1 (MeCN–H2O–DMSO =
79 : 20 : 1, v/v/v; pH = 7.5) was studied at 20 °C in the absence and pres-
ence of cysteine (100 equiv.) after the specified time periods (1, 10, 20,
30, 40, 50, 60, 120 min) with λex = 670 nm.
Fig. 2 (a) Absorption and (b) emission spectra (λex = 670 nm) of
probe 1 before (solid curve) and after (dashed curve) the addition of
cysteine. Final concentrations of 1 and cysteine are 2.0 × 10−5 M and
2.0 × 10−3 M, respectively, in MeCN–H2O–DMSO (79 : 20 : 1, v/v/v;
pH = 7.5). Data were collected before or 1 h after the addition of
cysteine at 20 °C.
Fig. 5 Fluorescence response of 20 μM probe 1 (MeCN–H2O–DMSO
= 79 : 20 : 1, v/v/v) at 755 nm upon reacting with cysteine in 1, 2, 10,
20, 40, 80, 120, 160, 200, 400, 800, 1200 μM concentrations after 1 h of
incubation at 20 °C. The excitation wavelength was 670 nm. The inner
panel displays the fluorescence enhancement of probe 1 toward cysteine
at 1, 2, 10, 20, 40, 80 μM.
intensity enhancement for cysteine, 3-aminothiophenol, and 1-
octylthiol, while slight enhancement for MeNH2, n-BuNH2,
aniline, glutamic acid, leucine and glutathione was observed.
Thus probe 1 exhibited the high selectivity toward thiols,
especially cysteine.
The response of probe 1 toward cysteine was carried out
(Fig. 4). After the addition of cysteine, a dramatic fluorescence
intensity increase was observed in a short time frame (<50 min).
No further remarkable enhancement was observed in an
extended reaction period up to 120 min.
The sensitivity of probe 1 was then studied by fluorescence
response towards various concentrations of cysteine (Fig. 5). A
distinct response of the probe 1 (20 μM) to cysteine in the con-
centration range of 1–120 μM was observed. When 20 equiv. of
cysteine (400 μM) was added, the enhancement of fluorescence
intensity reached the maximum in 1 h. Further increase of
cysteine concentration did not provide further enhancement of
Fig. 3 Selectivity of probe 1 toward various analytes. Relative fluor-
escence intensity of 20 μM probe 1 (MeCN–H2O–DMSO = 79 : 20 : 1,
v/v/v; pH = 7.5) was measured at 755 nm (λex = 670 nm) after incu-
bation at 20 °C for 1 h in the presence of 2 × 10−3 M (final concen-
trations) of analytes.
based on aza-BODIPY. These observations suggest that the
direct attachment of a DNBS group to the aza-BODIPY core
induced PET and quenched effectively the fluorophore’s fluor-
escence. A remarkable shift of the absorption (18 nm) and emis-
sion (21 nm) maxima between the probe
1 and the
corresponding cleavage product 2 was observed.
The selectivity of probe 1 towards various amino compounds
and biological thiols was investigated. As shown in Fig. 3, probe
1 was highly selective to thiols with remarkable fluorescence
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fluorescence intensity. Notably, the detection limit to cysteine
was found to be 5 × 10−7 M.
The effect of the pH on the fluorescence intensity and the
reactivity of probe 1 were examined. Probe 1 was very stable in
a range of pH 5–9, and no pH-dependent fluorescence change
was observed (see Fig. S1, ESI†). However, in the presence of
cysteine, the fluorescence intensity of probe 1 slightly varied
with pH value (pH = 5–9),13a and probe 1 exhibited the most
sensitive response under weakly basic conditions (pH = 8) as a
result of the ionization of cysteine.19
In conclusion, we have developed a new fluorescent turn-on
NIR probe based on the aza-BODIPY dye. Probe 1 is weakly
fluorescent. Cleavage of DNBS with thiols releases the aza-
BODIPY fluorophore 2 with concurrent fluorescence in the NIR
region (λem = 755 nm, Φf = 0.14). It is highly selective towards
cysteine and the detection is rapid. An increase in the of concen-
tration of cysteine affords a quicker and more dramatic response,
and the detection limit to cysteine reaches 5 × 10−7 M. Probe 1
can be applied to neutral conditions with a pH range (7–8) that
is compatible with most biological applications. Importantly, the
remarkable shift of the absorption (18 nm) and emission (21 nm)
maxima between probe 1 and the corresponding cleavage
product 2 is observed to distinctly raise the detection sensitivity.
Efforts to create a new fluorescent turn-on NIR-emitting thiol
probe based on a NIR dye with high fluorescence contrast are
now ongoing in our lab.
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This work was supported by the National Natural Science Foun-
dation of China (20872026), National Basic Research Program
of China (973 Program) No. 2010CB912600, Shanghai Pujiang
Talent Plan Project (09PJD008), and Sino Swiss Science and
Technology Cooperation (SSSTC, EG 30-032010).
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This journal is © The Royal Society of Chemistry 2012